Biomedical subjects

S H Wilson

Publications and source records attributed to S H Wilson.

At least 109 records · Page 6Linked to original sources

PubMed1996

Activation of the human DNA polymerase beta promoter by a DNA-alkylating agent through induced phosphorylation of cAMP response element-binding protein-1.

Treatment of cells with the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induces expression of the endogenous mammalian DNA polymerase beta (beta-pol) gene and of the cloned promoter in transient expression studies. The lone cAMP response element (CRE) in the core promoter, along with functional protein kinase A, is critical for the MNNG-induced up-regulation. Recently, we described a kinetic mechanism for transcriptional regulation of the beta-pol promoter in vitro and found that CRE-binding protein (CREB) from MNNG-treated cells differentially up-regulates the promoter by stimulating formation of closed preinitiation complex (RPc). Here, using a CRE-dependent chimeric beta-pol promoter, we purified the RPc assembled with nuclear extract from MNNG-treated and control HeLa cells. Comparison of proteins in the purified RPc samples revealed that the MNNG induction is associated with a strong increase in the Ser133-phosphorylated form of recombinant CREB (CREB-1). CREB depletion of the nuclear extracts diminished transcriptional activity, and addition of purified Ser133-phosphorylated CREB-1 restored activity, whereas unphosphorylated CREB-1 did not. Addition of phosphorylated CREB-1 to the control cell extract mimicked the MNNG-induced up-regulation of transcriptional activity. These results indicate that phosphorylation of CREB-1 is the probable mechanism of activation of the beta-pol promoter after treatment of cells with the DNA-alkylating agent MNNG.

Alkylating Agents↗

PubMed1996

Evidence for an imino intermediate in the DNA polymerase beta deoxyribose phosphate excision reaction.

A recent study demonstrated that rat DNA polymerase beta (beta-pol) releases 5'-deoxyribose phosphate (dRP) termini from preincised apurinic/apyrimidinic DNA, a substrate generated during certain types of base excision repair. This catalytic activity resides within the amino-terminal, 8-kDa domain of beta-pol and occurs via beta-elimination as opposed to hydrolysis (Matsumoto, Y., and Kim, K. (1995) Science 269, 699-702). The latter finding suggested that the dRP excision reaction might proceed via an imine intermediate. In order to test this hypothesis, we attempted to trap beta-pol on preincised apurinic/apyrimidinic DNA using NaBH4 as the reducing agent. Both 8-kDa domain-DNA and intact beta-pol-DNA complexes were detected and identified by autoradiography coupled to immunoblotting. Our results indicate that the chemical mechanism of the beta-pol dRpase reaction does proceed through an imine enzyme-DNA intermediate and that the active site residue responsible for dRP release must therefore contain a primary amine.

Amino Acid Sequence↗

PubMed1996

Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis.

Base excision repair (BER) is a cellular defense mechanism repairing modified bases in DNA. Recently, a G:U repair reaction has been reconstituted with several purified enzymes from Escherichia coli (Dianov, G., and Lindahl, T.(1994) Curr. Biol. 4, 1069-1076). Using bovine testis crude nuclear extract, we have shown that G:U is repaired efficiently in vitro, and DNA polymerase beta (beta-pol) is responsible for the single nucleotide gap-filling synthesis (Singhal, R. K., Prasad, R., and Wilson, S. H.(1995) J. Biol. Chem. 270, 949-957). To investigate potential interaction of beta-pol with other BER protein(s), we developed affinity chromatography matrices by cross-linking purified rat beta-pol or antibody against beta-pol to solid supports. Crude nuclear extract from bovine testis was applied to these affinity columns, which were then extensively washed. Proteins that bound specifically to the affinity columns were co-eluted in a complex with beta-pol. This complex had a molecular mass of approximately 180 kDa and was able to conduct the complete uracil-initiated BER reaction. The BER complex contained both beta-pol and DNA ligase I. An antibody to beta-pol was able to shift the complex in sucrose gradients to a much larger molecular mass (>300 kDa) that again contained both beta-pol and DNA ligase I. Furthermore, DNA ligase I and beta-pol were co-immunoprecipitated from the testis nuclear extract with anti beta-pol IgG. Thus, we conclude that beta-pol and DNA ligase I are components of a multiprotein complex that performs BER.

Animals↗

PubMed1996

DNA polymerases alpha and beta are required for DNA repair in an efficient nuclear extract from Xenopus oocytes.

Xenopus oocytes and an oocyte nuclear extract efficiently repair the bulky DNA lesions cyclobutane pyrimidine dimers,(6-4) photoproducts, and N-acetoxy-2-aminofluorene (AAF) adducts by an excision repair mechanism. Nearly all (>95%) of the input damaged DNA was repaired within 5 h in both injected cells and extracts with no significant incorporation of label into control undamaged DNA. Remarkably, more than 10(10) cyclobutane pyrimidine dimers or(6-4) photoproducts are repaired/nuclei. The extracts are free from nuclease activity, and repair is independent of exogenous light. Both the high efficiency and DNA polymerase requirements of this system appear to be different from extracts derived from human cells. We demonstrated a requirement for DNA polymerases alpha and beta in repair of both photoproducts and AAF by inhibiting repair with several independent antibodies specific to either DNA polymerases alpha or beta and then restoring repair by adding the appropriate purified polymerase. Repair is inhibited by aphidicolin at concentrations specific for blocking DNA polymerase alpha and dideoxynucleotide triphosphates at concentrations specific for inhibiting DNA polymerase beta.

Animals↗

PubMed1996

Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase beta.

In the crystal structure of a substrate complex, the side chains of residues Asn279, Tyr271, and Arg283 of DNA polymerase beta are within hydrogen bonding distance to the bases of the incoming deoxynucleoside 5'-triphosphate (dNTP), the terminal primer nucleotide, and the templating nucleotide, respectively (Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H., and Kraut, J. (1994) Science 264, 1891-1903). We have altered these side chains through individual site-directed mutagenesis. Each mutant protein was expressed in Escherichia coli and was soluble. The mutant enzymes were purified and characterized to probe their role in nucleotide discrimination and catalysis. A reversion assay was developed on a short (5 nucleotide) gapped DNA substrate containing an opal codon to assess the effect of the amino acid substitutions on fidelity. Substitution of the tyrosine at position 271 with phenylalanine or histidine did not influence catalytic efficiency (kcat/Km) or fidelity. The hydrogen bonding potential between the side chain of Asn279 and the incoming nucleotide was removed by replacing this residue with alanine or leucine. Although catalytic efficiency was reduced as much as 17-fold for these mutants, fidelity was not. In contrast, both catalytic efficiency and fidelity decreased dramatically for all mutants of Arg283 (Ala > Leu > Lys). The fidelity and catalytic efficiency of the alanine mutant of Arg283 decreased 160- and 5000-fold, respectively, relative to wild-type enzyme. Sequence analyses of the mutant DNA resulting from short gap-filling synthesis indicated that the types of base substitution errors produced by the wild-type and R283A mutant were similar and indicated misincorporations resulting in frequent T.dGTP and A.dGTP mispairing. With R283A, a dGMP was incorporated opposite a template thymidine as often as the correct nucleotide. The x-ray crystallographic structure of the alanine mutant of Arg283 verified the loss of the mutated side chain. Our results indicate that specific interactions between DNA polymerase beta and the template base, but not hydrogen bonding to the incoming dNTP or terminal primer nucleotide, are required for both high catalytic efficiency and nucleotide discrimination.

Base Sequence↗

PubMed1996

Role of the "helix clamp" in HIV-1 reverse transcriptase catalytic cycling as revealed by alanine-scanning mutagenesis.

Residues 259-284 of HIV-1 reverse transcriptase exhibit sequence homology with other nucleic acid polymerases and have been termed the "helix clamp" (Hermann, T., Meier, T., Gotte, M., and Heumann, H. (1994) Nucleic Acids Res. 22, 4625-4633), since crystallographic evidence indicates these residues are part of two alpha-helices (alpha H and alpha I) that interact with DNA. Alanine-scanning mutagenesis has previously demonstrated that several residues in alpha H make important interactions with nucleic acid and influence frameshift fidelity. To define the role of alpha I (residues 278-286) during catalytic cycling, we performed systematic site-directed mutagenesis from position 277 through position 287 by changing each residue, one by one, to alanine. Each mutant protein was expressed and, except for L283A and T286A, was soluble. The soluble mutant enzymes were purified and characterized. In contrast to alanine mutants of alpha H, alanine substitution in alpha I did not have a significant effect on template.primer (T.P) binding as revealed by a lack of an effect on Km, T.P, Ki for 3'-azido-2',3'-dideoxythymidine 5'-triphosphate, koff, T.P and processivity. Consistent with these observations, the fidelity of the mutant enzymes was not influenced. However, alanine mutagenesis of alpha I lowered the apparent activity of every mutant relative to wild-type enzyme. Titration of two mutants exhibiting the lowest activity with T.P (L282A and R284A) demonstrated that these mutant enzymes could bind T.P stoichiometrically and tightly. In contrast, active site concentrations determined from "burst" experiments suggest that the lower activity is due to a smaller populations of enzyme bound productively to T.P. The putative electrostatic interactions between the basic side chains of the helix clamp and the DNA backbone are either very weak or kinetically silent. In contrast, interactions between several residues of alpha H and the DNA minor groove, 3-5 nucleotides from the 3'-primer terminus, are suggested to be critical for DNA binding and fidelity.

Alanine↗

PubMed1996

Three-dimensional solution structure of the N-terminal domain of DNA polymerase beta and mapping of the ssDNA interaction interface.

DNA polymerase beta (beta-Pol) consists of an N-terminal ssDNA binding domain with deoxyribose phosphodiesterase activity and a C-terminal domain with nucleotidyltransferase activity. The solution structure of the cloned N-terminal domain of beta-Pol has been determined by multidimensional heteronuclear NMR using experimental restraints that included 1030 distances based on analysis of NOE connectivities, 68 phi, chi 1, and chi 2 torsion angles based on analysis of couplings, and 22 hydrogen bonds. Hydrogen bonds were assessed only within helices by the absence of saturation transfer from water at pH 6.7, by NOEs and JNH alpha couplings indicative of well-structured helices, and by 13C alpha chemical shifts characteristic of helices. The root mean square deviation for heavy backbone atoms within the helices was 0.64 A in 55 structures. The solution structure of the N-terminal domain is formed from four helices packed as two antiparallel pairs crossing at 50 degrees in a V-like shape. The domain binds p(dT)8, a template analogue, as a 1:1 complex in 100 mM NaCl (KD = 10 microM). Analysis of the binding equilibria at increasing NaCl concentrations indicated that ionic contacts contribute to the complex. The binding interaction was mapped to one face of the domain by characterizing backbone 1H and 15N chemical shift changes. Assigned intermolecular NOEs from 2D NOESY support the assessment of the binding interface. The structure that forms the interaction surface includes an antiparallel helix-3-turn-helix-4 motif and residues adjacent to an omega-type loop connecting helix-1 and helix-2. Sites appropriate for nucleotide contact on the structure are described. The mapped interaction interface for a ssDNA template is the first described for a DNA polymerase.

Amino Acid Sequence↗

PubMed1996

dNTP binding site in rat DNA polymerase beta revealed by controlled proteolysis and azido photoprobe cross-linking.

Mild proteolysis of rat DNA polymerase beta (beta-pol) generates an N-terminal 8 kDa domain and a C-terminal 31 kDa domain; the 31 kDa domain is degraded to 6 and 27 kDa fragments by further proteolysis [Kumar, A., Widen, S.G., Williams, K.R., Kedar, P., Karpel, R.L., & Wilson S.H. (1990) J. Biol. Chem. 265, 2124-2131]. In the present study, we found that more vigorous trypsin digestion of the 27 kDa fragment of beta-pol produces 10 and 12 kDa subdomains. Thus, rat beta-pol has four distinct proteolytic fragments of 8, 6, 10, and 12 kDa, extending from the N-terminus to the C-terminus, respectively. To map the location of the dNTP binding site(s), intact beta-pol was photoaffinity labeled with 8-azido-ATP or 5-azido-dUTP in presence or absence of competitor dNTP (dATP). The labeled enzyme was subjected to controlled proteolysis, and the resulting labeled peptides were separated and sequenced. Competition with dATP showed that three regions of beta-pol in solution combine to form the dNTP binding pocket as follows: residues 4-40 of the 8 kDa domain; residues 142-206 of the 10 kDa subdomain; and residues 263-280 of the 12 kDa subdomain (alpha-helices M and N). These results are discussed in light of the recent crystal structure of dATP bound to rat beta-pol.

Adenosine Triphosphate↗

PubMed1996

Purification and characterization of a DNA polymerase beta promoter initiator element-binding transcription factor from bovine testis.

A low-abundance DNA-binding protein for the DNA polymerase beta (beta-pol) promoter initiator element was purified from bovine testis. The transcriptional initiator element (Inr) of the mammalian beta-pol promoters characterized is highly conserved, and the bovine beta-pol promoter Inr has the sequence -11CAGAGGCGGCCATTGTT+6. The purified initiator element-binding protein (Inr-BP) binds with high affinity to an oligonucleotide corresponding to the beta-pol promoter Inr (Kd = 5 pM), and increasing ionic strength decreases stability of the protein-DNA complex. Mutational analysis of the Inr shows that the purified Inr-BP binds with sequence specificity to the sequence CCAT at -2 to +2 of the Inr, but that seven residues on the 5' side and three residues on the 3' side of the CCAT sequence are required also. Using an in vitro transcription assay with HeLa cell nuclear extract, we find that the endogenous Inr-BP is required for transcriptional activity of the beta-pol promoter; addition of purified Inr-BP restores activity to the nuclear extract depleted in Inr-BP by affinity chromatography. These results, based upon the sequence specificity for DNA binding, indicate that Inr-BP is a YY1-like protein and suggest that it is a required transcription factor in beta-pol gene expression.

Animals↗

PubMed1996

Requirement of mammalian DNA polymerase-beta in base-excision repair.

Synthesis of DNA by DNA polymerase-beta is distributive on single-stranded DNA templates, but short DNA gaps with a 5' PO4 in the gap are filled processively to completion. In vitro studies have suggested a role of beta-polymerase in different types of DNA repair. However, the significance of these studies to the in vivo role of beta-polymerase has remained unclear. Because genetic studies are essential for determining the physiological role of a gene, we established embryonic fibroblast cell lines homozygous for a deletion mutation in the gene encoding DNA polymerase-beta. Extracts from these cell lines were found to be defective in uracil-initiated base-excision repair. The beta-polymerase-deleted cells are normal in viability and growth characteristics, although they exhibit increased sensitivity to monofunctional DNA-alkylating agents, but not to other DNA-damaging agents. Both the deficiency in base-excision repair and hypersensitivity to DNA-alkylating agents are rescued following stable transfection with a wild-type beta-polymerase minitransgene. These studies demonstrate that beta-polymerase functions specifically in base-excision repair in vivo.

Animals↗

PubMed1996

Identification of novel mRNA isoforms for human DNA polymerase beta.

Recently, we reported the organization of the thirteen exons of the human DNA polymerase beta (beta-pol) gene and the sequences of the exon-intron junctions. Splice variants of human beta-pol mRNA have been postulated to be related to cancer development. Here, we report the characterization of isoforms of human beta-pol mRNA in different cells by reverse transcription polymerase chain reaction (RT-PCR). DNA sequence analysis of RT-PCR products revealed eight alternative splicing mRNA isoforms in the brain cancer cell line, SK-N-MC. These various isoforms were consistent with alternative splicing of four exons (II, IV, V, and VI) and with a 105-nucleotide insertion (exon alpha) between exons VI and VII. We also found an isoform with a 19-nucleotide sequence inserted into the exon IV and V junction, which resulted from usage of a different 3' splice site. Seven of the isoforms resulted in truncated open reading frame (ORF); five corresponded to deduced peptide of amino acids 1-20 of beta-pol and two corresponded to amino acids 1-60 of beta-pol. Only one of the right mRNA isoforms, that with the exon alpha insertion, was in-frame with the entire wild-type ORF resulting in a deduced protein of 370 residues, compared with the wild-type protein of 335 residues and 39 kD. This longer ORF was shown to be capable of encoding a beta-pol protein, larger than wild-type beta-pol, that cross-reacted with beta-pol antibody and exhibited beta-pol enzymatic activity. The mRNA isoform with the exon alpha insertion was not tumor specific because it as detected in low abundance in all cells tested, except the colon cell line CCD18 Co where the isoform was absent. The genomic location of exon alpha is in intron VI, 990 bp upstream of exon VII and flanked by consensus splice sites. Thus, this 105-bp genomic sequence is a beta-pol exon present in a low-abundance beta-pol mRNA isoform capable of encoding an approximately 42-kD beta-pol.

Alternative Splicing↗

PubMed1996

A comparison of gross pathology, histopathology, and mycobacterial culture for the diagnosis of tuberculosis in elk (Cervus elaphus).

Using the isolation of Mycobacterium bovis as the reference standard, this study evaluated the sensitivity, specificity and kappa statistic of gross pathology (abattoir postmortem inspection), histopathology, and parallel or series combinations of the two for the diagnosis of tuberculosis in 430 elk and red deer. Two histopathology interpretations were evaluated: histopathology I, where the presence of lesions compatible with tuberculosis was considered positive, and histopathology II, where lesions compatible with tuberculosis or a select group of additional possible diagnoses were considered positive. In the 73 animals from which M. bovis was isolated, gross lesions of tuberculosis were most often in the lung (48), the retropharyngeal lymph nodes (36), the mesenteric lymph node (35), and the mediastinal lymph nodes (16). Other mycobacterial isolates included: 11 M. paratuberculosis, 11 M. avium, and 28 rapidly growing species or M. terrae complex. The sensitivity estimates of gross pathology and histopathology I were 93% (95% confidence limits [CL] 84.97%) and 88% [CL 77.94%], respectively, and the specificity of both was 89% [CL 85.92%]). The sensitivity and specificity of histopathology II were 89% (CL 79.95%) and 77% (CL 72.81%), respectively. The highest sensitivity estimates (93-95% [CL 84.98%]) were obtained by interpreting gross pathology and histopathology in parallel (where an animal had to be positive on at least one of the two, to be classified as combination positive). The highest specificity estimates (94-95% [CL 91-97%] were generated when the two tests were interpreted in series (an animal had to be positive on both tests to be classified as combination positive). The presence of gross or microscopic lesions showed moderate to good agreement with the isolation of M. bovis (Kappa = 65-69%). The results showed that post-mortem inspection, histopathology and culture do not necessarily recognize the same infected animals and that the spectra of animals identified by the tests overlaps.

Abattoirs↗

PubMed1995

The bovine DNA polymerase beta promoter: cloning, characterization and comparison with the human core promoter.

The core promoter of the human DNA polymerase beta (beta Pol)-encoding gene (POL beta) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755-12763]. To study the homologous promoter (ppol beta) in a bovine system, we cloned and characterized the 5'-flanking region of the bovine gene (pol beta). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5'-flanking region was isolated from a testis library in bacteriophage lambda EMBL3. S1 nuclease mapping and primer extension analysis of the 5'-end of the pol beta mRNA identified the major transcription start point (tsp), which is located 142-bp 5' of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5'-deletion mutants demonstrated that a fragment of only 91-bp 5' of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOL beta). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Sp1-binding sites, and the spacing separating them.

Animals↗

PubMed1995

Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-pol is down-regulated in the antisense cell line. Such a sensitization is anticipated only where beta-pol is rate-limiting in a DNA repair pathway. A number of agents were tested: cis-diamminedichloroplatinum II (cisplatin); 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU); ionizing radiation and the radio-mimetic drug bleomycin; the bifunctional alkylating agents nitrogen mustard and L-phenylalanine mustard (melphalan); the monofunctional alkylating agent methyl methane sulfonate (MMS) and ultraviolet (UV) radiation. In the cases of cisplatin and UV radiation, a significant enhancement of cytotoxicity was observed. Damage as a result of both of these agents is thought to be repaired by the nucleotide excision repair (NER) pathway. The results suggest that, in this cell line, beta-pol is involved in and is rate-limiting in NER. We propose that down-regulation of beta-pol by antisense approaches might be used to enhance the cytotoxic effects of cisplatin and other DNA damaging chemotherapeutic agents.

3T3 Cells↗

PubMed1995

Reduced frameshift fidelity and processivity of HIV-1 reverse transcriptase mutants containing alanine substitutions in helix H of the thumb subdomain.

We have analyzed two human immunodeficiency virus (HIV-1) reverse transcriptase mutants of helix H in the thumb subdomain suggested by x-ray crystallography to interact with the primer strand of the template-primer. These enzymes, G262A and W266A, were previously shown to have greatly elevated dissociation rate constants for template-primer and to be much less sensitive to inhibition by 3'-azidodeoxythymidine 5'-triphosphate. Here we describe their processivity and error specificity. The results reveal that: (i) both enzymes have reduced processivity and lower fidelity for template-primer slippage errors, (ii) they differ from each other in sequence-dependent termination of processive synthesis and in error specificity, and (iii) the magnitude of the mutator effect relative to wild-type enzyme for deletions in homopolymeric sequences decreases as the length of the run increases. Thus amino acid substitutions in a subdomain thought to interact with the duplex template-primer confer a strand slippage mutator phenotype to a replicative DNA polymerase. This suggests that interactions between specific amino acids and the primer stem at positions well removed from the active site are critical determinants of processivity and fidelity. These effects, obtained in aqueous solution during catalytic cycling, are consistent with and support the existing crystallographic structural model.

Alanine↗

PubMed1995

Phorbol ester abrogates up-regulation of DNA polymerase beta by DNA-alkylating agents in Chinese hamster ovary cells.

Mammalian DNA polymerase beta (beta-pol), a DNA repair polymerase, is known to be constitutively expressed in cultured cells, but treatment of cells with the DNA-alkylating agents MNNG or methyl methanesulfonate has been shown to up-regulate beta-pol mRNA level. To further characterize this response, we prepared a panel of monoclonal antibodies and used one of them to quantify beta-pol in whole cell extracts by immunoblotting. We found that treatment of Chinese hamster ovary cells with either DNA-alkylating agent up-regulated the beta-pol protein level 5-10-fold. This induction appeared to be secondary to DNA alkylation, as induction was not observed with a genetically altered cell line overexpressing the DNA repair enzyme O6-methylguanine-methyltransferase. We also found that 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment of wild type Chinese hamster ovary cells increased expression of beta-pol protein (approximately 10-fold). Any interrelationship between this TPA response and the DNA-alkylation response was studied by treatment with combinations of MNNG and TPA. The beta-pol up-regulation observed with MNNG treatment was abrogated by TPA, and conversely the up-regulation observed with TPA treatment was abrogated by MNNG.

Alkylating Agents↗

PubMed1995

Specific inhibition of DNA polymerase beta by its 14 kDa domain: role of single- and double-stranded DNA binding and 5'-phosphate recognition.

DNA polymerase beta (beta-polymerase) has been implicated in short-patch DNA synthesis in the DNA repair pathway known as base excision repair. The native 39 kDa enzyme is organized into four structurally and functionally distinct domains. In an effort to examine this enzyme as a potential therapeutic target, we analyzed the effect of various beta-polymerase domains on the activity of the enzyme in vitro. We show that the 14 kDa N-terminal segment of beta-polymerase, which binds to both single- and double-stranded DNA, but lacks DNA polymerase activity, inhibits beta-polymerase activity in vitro. Most importantly, the 8, 27 and 31 kDa domains of beta-polymerase do not inhibit beta-polymerase activity, demonstrating that the inhibition by the 14 kDa domain is specific. The inhibition of beta-polymerase activity in vitro is abolished by increasing the concentrations of both of the substrates (template-primer and deoxynucleoside triphosphate). In contrast, an in vitro base excision repair assay is inhibited in a domain specific manner by the 14 kDa domain even in the presence of saturating substrates. The inhibition of beta-polymerase activity by the 14 kDa domain appears specific to beta-polymerase as this domain does not inhibit either mammalian DNA polymerase alpha or Escherichia coli polymerase I (Klenow fragment). These data suggest that the 14 kDa domain could be used as a potential inhibitor of intracellular beta-polymerase and that it may provide a means for sensitizing cells to therapeutically relevant DNA damaging agents.

Base Sequence↗

PubMed1995

Human immunodeficiency virus type 1 reverse transcriptase. 3'-Azidodeoxythymidine 5'-triphosphate inhibition indicates two-step binding for template-primer.

Human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) catalyzes DNA synthesis by an ordered sequential mechanism. After template-primer (T.P) binds to free enzyme, the deoxynucleoside triphosphate to be incorporated binds to the RT and T.P binary complex (RTT.P). After incorporation of the bound nucleotide, catalytic cycling is limited either by a conformational change (for processive synthesis) or release of the enzyme from the extended T.P (for single-nucleotide incorporation). To explore cycling through these alternate rate-limiting steps, we determined kinetic parameters for single-nucleotide incorporation by HXB2R HIV-1 RT with chain-terminating nucleotide substrates 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and dideoxythymidine triphosphate on a homopolymeric T.P system, poly(rA)-oligo(dT)16. Inhibition of processive deoxythymidine monophosphate incorporation by these chain-terminating substrates was also examined. Because AZTTP is a substrate, its Km should be equivalent to Ki, and since Km for AZTTP should be influenced by the dissociation rate constant for RTT.P, we examined the effect of altering RTT.P dissociation on AZTTP kinetic parameters. The dissociation rate constant was modulated by making use of different T.P substrates, viral sources of RT, and a mutant RT altered at a residue that perturbs T.P binding. As expected from earlier work, the time course of AZTMP incorporation on poly(rA)-oligo(dT)16 was biphasic, with a burst followed by a slower steady-state phase representing kcat (0.42 min-1) which was similar to the rate constant for RTT.P dissociation. Additionally, Km for AZTTP (110 nM) was lower than its equilibrium dissociation constant (1200 nM). AZTTP inhibition (Ki,AZTTP) of processive dTMP incorporation and incorporation of a single nucleotide were similar. However, a simple correlation between the RTT.P dissociation rate constant and Ki,AZTTP was not observed. These results indicate that a simple ordered model for single-nucleotide incorporation is inadequate and that different forms of RTT.P exist which can limit catalysis. The results are discussed in the context of a two-step binding reaction for T.P where the binary RTT.P complex undergoes an isomerization before binding of the deoxynucleotide substrate.

Antiviral Agents↗